Phosphorylation of human oxoguanine DNA glycosylase (alpha-OGG1) modulates its function - PubMed (original) (raw)

Phosphorylation of human oxoguanine DNA glycosylase (alpha-OGG1) modulates its function

Jingping Hu et al. Nucleic Acids Res. 2005.

Abstract

Oxoguanine DNA glycosylase (OGG1) initiates the repair of 8-oxoguanine (8-oxoG), a major oxidative DNA base modification that has been directly implicated in cancer and aging. OGG1 functions in the base excision repair pathway, for which a molecular hand-off mechanism has been proposed. To date, only one functional and a few physical protein interactions have been reported for OGG1. Using the yeast two-hybrid system and a protein array membrane, we identified two novel protein interactions of OGG1, with two different protein kinases: Cdk4, a serine-threonine kinase, and c-Abl, a tyrosine kinase. We confirmed these interactions in vitro using recombinant proteins and in vivo by co-immunoprecipitation from whole cell extracts. OGG1 is phosphorylated in vitro by Cdk4, resulting in a 2.5-fold increase in the 8-oxoG/C incision activity of OGG1. C-Abl tyrosine phosphorylates OGG1 in vitro; however, this phosphorylation event does not affect OGG1 8-oxoG/C incision activity. These results provide the first evidence that a post-translational modification of OGG1 can affect its catalytic activity. The distinct functional outcomes from serine/threonine or tyrosine phosphorylation may indicate that activation of different signal transduction pathways modulate OGG1 activity in different ways.

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Figures

Figure 1

Figure 1

Physical interaction between OGG1 and Cdk4. (A) Yeast strain AH109 [Matchmaker™ Gal4 Two-hybrid system 3 (Clontech)] was transformed with pGBKT7-_OGG1_-α and p34SEI-1 or empty Gal4-AD vector. The yeast were cultured in high-stringency selective meduim and allowed to grow for 1 week. (B) Recombinant his-tagged OGG1 (OGG1-6his) and GST-tagged Cdk4 (Santa Cruz Biotech.) were separated on 12% Tris–glycine polyacrylamide gels. One gel was stained with Coomassie blue and the others were electro-transferred to PVDF membranes. The membranes were then incubated with either purified OGG1-6his (+OGG1) or BSA (+BSA) and probed with anti-OGG1 antibody, as described in Materials and Methods. Lanes 1 and 2 contain 800 and 400 ng Cdk4-GST protein; lane 3 contains 450 ng OGG1-6his in the Coomassie gel and 10 ng in the immunoblots.

Figure 2

Figure 2

OGG1 and Cdk4 interact in vivo. HEK293 cells were transfected with FLAG-tagged OGG1 plasmid or empty vector. After 2 weeks selection with G418 (Invitrogen), whole cell extracts were prepared from the pool of cells as described in Materials and Methods. A monoclonal antibody, anti-FLAG-M2 agarose (Sigma), was used to immunoprecipitate FLAG-tagged OGG1. The immunoprecipitates were separated on 12% Tris-glycine PAGE gels and analyzed by western blot with the indicated antibodies.

Figure 3

Figure 3

In vitro phosphorylation of OGG1 by Cdk4. (A) Recombinant OGG1-6his (62, 125, 250, 500 and 1000 ng), purified from insect cell (Sf9), was immunobloted with an anti-phosphoserine antibody. (B) An aliquot of 100 ng of purified OGG1 was incubated with either active immunoprecipitated Cdk4 beads (lanes 1 and 3) or heat-inactivated immunoprecipitated Cdk4 beads (lane 2) in the presence of [γ-32P]ATP. One sample (lane 1) was then subjected to PP1 treatment after Cdk4 phosphorylation. The phosphorylated proteins were analyzed by autoradiography, followed by electro-blotting of the same gel to PVDF membrane, and western blot analysis with the indicated antibodies.

Figure 4

Figure 4

OGG1 phosphorylation by Cdk4 increases 8-oxoG strand cleavage and base release activities. 10 ng of purified OGG1-α-6his was first incubated with PP1 to ensure complete dephosphorylation. The proteins were then incubated with immunoprecipitated Cdk4, or heat-inactivated Cdk4 as a negative control. (A) Serial dilutions of OGG1-α-6his (0.25, 0.5, 1, 2 and 4 ng) were analyzed for 8-oxoG strand cleavage activity measurement, as described in Materials and Methods. (B) Similar OGG1-α-6his concentrations were analyzed for base release activity. The graphs on the right side of each panel present the average ±SEM of at least two independent replicates. Asterisks, significantly higher than inactive Cdk4 at P < 0.05.

Figure 5

Figure 5

PKC phosphorylates OGG1 in vitro but does not change its incision activity. (A) An aliquot of 50–100 ng of recombinant OGG1-6his was incubated with or without 5 ng PKC (Promega) in the presence of [γ-32P]ATP. The reactions were separated by SDS–PAGE and analyzed by autoradiography (top panel) and western blotting (bottom panel). (B) An aliquot of 0.25–6 ng of purified OGG1-α-6his was incubated with or without 5 ng PKC and 8-oxoG incision activity was measured as described in Materials and Methods. The graph presents the average ±SEM of two independent replicates.

Figure 6

Figure 6

c-Abl physically interacts with and phosphorylates OGG1 in vitro. (A) An aliquot of 25, 50 and 100 ng of Cdk4, XRCC1, c-Abl, WRN and hOGG1 were applied onto the DiscoverLight™ Protein-array membrane. The membrane was processed and developed as described in the Materials and Methods. As positive controls, we blotted 1 μl of 293-FLAG-OGG1 lysate and a horseradish peroxidase control provided by the manufacturer. (B) Purified OGG1-α-6his was incubated with either GST (lane 1) or GST-c-Abl fusion proteins (inactivated in lane 2, active in lane 3). GST-pull down was performed as described in Materials and Methods. The proteins were then detected by immunoblot. OGG1-α-6his was loaded in lane 4 as a positive control. (C) Purified OGG1-6his was incubated with 100 ng full-length c-Abl either in the presence (lanes 2–4) or absence of ATP (lanes 5–7) to look at tyrosine phosphorylation. The proteins were analyzed by immunoblot with anti-phosphotyrosine (middle panel) followed by anti-OGG1 (top panel) and anti-c-Abl (bottom panel) antibodies.

Figure 7

Figure 7

c-Abl phosphorylation of OGG1 does not change 8-oxoG incision. (A) Purified OGG1-6his protein (12.5, 25 and 50 ng) was incubated with full-length c-Abl (100 ng in each reaction), either active (lanes 2–4, allowing tyrosine phosphorylation) or heat-inactivated (lanes 5–7, unphosphorylated). The proteins were then analyzed by western blotting with the specified antibodies. (B) Purified OGG1-6his (0.25–12 ng) was incubated with 100 ng active (lanes 3–8) or heat-inactivated (lanes 9–14) c-Abl and then used for an 8-oxoG incision activity assay, as described in Materials and Methods. The graph presented shows the average ±SEM of at least two independent replicates.

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